S. aureus is a prominent and successful human pathogen. It is able to cause a wide range of disease, affecting any and all organs. Major proponents of disease are surface associated and secreted proteins. While many virulence determinants have been identified, the mechanism(s) by which they are secreted by the bacterium are not well understood. Previous research has identified that S. aureus cell wall proteins are deposited to two separate, but distinct locations. This differential localization is determined by the presence or absence of a motif within the signal sequence, YSIRK G/S. Cell wall proteins containing this domain are delivered to the bacterial cross wall, the area of active cell division, while those proteins without the motif are trafficked to the cell periphery. It is unknown what cellular machinery governs this unique pathway. The hypothesis is that there are two distinct pathways for protein secretion. Specific cellular proteins are able to differentiate between exported proteins which do and do not contain the YSIRK motif to transport them to their respective location. This proposal will utilize chemical and transposon mutagenesis to identify genes required for protein secretion and cell wall deposition. Of particular importance is the identification of mutants that preferentially affect one pathway rather than global defects. Chemical mutagenesis has already been performed, but will be expanded, and mutants will be phenotypically evaluated to identify the precise location of the secretion defect. Mutants can then be categorized to subsequently help create a mechanistic understanding of the genes that are required for proper protein secretion, either into the extracellular milieu or bound to the cell wall. Chemically derived mutants will be mapped and transposon insertions will be confirmed. An understanding of protein secretion in S. aureus is essential to better control its disease. Due to the essential nature of secreted and cell wall associated proteins to pathogenesis, identification of the mechanism driving this process can identify novel therapeutic targets. Due to the increase in antibiotic resistance and overall spread of infections of recent years, it is imperative to develop new pharmacological treatments.